Construction of Hydrogen Peroxide Responsive Fluorescent Nanoprobes for Circulating Tumor Cells Detection

Author:Li Chun Zuo

Supervisor:zhu xin yuan li pei yong


Degree Year:2018





Circulating tumor cells(CTC)that are shed from primary tumor sites play an important role in the early diagnosis and curative effect assessment of tumor.On the other hand,hydrogen peroxide(H2O2)is the second messenger in cells and plays a critical role in regulating cell proliferation,differentiation and migration.Meanwhile,angiogenesis,oxidative stress,aging and cancer developing are closely related to high concentration of H2O2.It is crucial to distingwish the signals of H2O2 and take advantage of high H2O2 concentration for CTC detection in timely diagnosis of malignant tumor.Recently,a series of smart nanosystems response to over-expressed H2O2 in tumorous macroenvionment.They are applied in dianostic imaging,tumor hypoxia symptoms,photodynamic therapy,drug controlled release and so on.Therefore,developing novel smart H2O2–responsive nanosystems for cancer diagnosis is still a hot research topic in the nanomedicine field.In this dissertation,we have constructed a series of H2O2-responsive fluorescent nanoprobes to detect the H2O2 level in circulating tumor cells,and it is helpful for early diagnosis of tumors.The main research contents and conclusions in this dissertation are summarized as below:1.“Turn on”hydrogen peroxide responsive fluorescent nanoprobe and its circulating tumor cell identification and colorectal cancer diagnosisDue to the high heterogeneity of tumor cells and the epithelial-mesenchymal transition(EMT)effect,traditional circulating tumor cell(CTC)assays are most likely to result in false-positive or false-negative results.Therefore,a hydrogen peroxide responsive fluorescent nanoprobe(GC-Cou-Bpin)based on a coumarin molecule was constructed.Hydrophobic coumarin-boric acid ester was grafted to the side chain of hydrophilic glycol chitosan through amidation reaction to prepare the amphiphilic graft polymer,which can self-assemble in water to form fluorescent nanoprobe.This nanoprobe can quickly enter cells and react with intracellular hydrogen peroxide.Herein,its blue fluorescent signal was markedly enhanced by the removal of boronate protecting groups by intracellular high levels of hydrogen peroxide.In vitro cell evaluation confirmed that the GC-Cou-Bpin fluorescent nanoprobe can effectively respond to and image hydrogen peroxide in colon cancer cells.The GC-Cou-Bpin fluorescent nanoprobe is further applied to the detection of circulating tumor cells in colon cancer patients with different malignancies.The clinical experimental results demonstrated that the GC-Cou-Bpin nanoprobe could effectively overcome the false-negative findings due to the low expression of cytokeratin 19(CK19)in circulating tumor cells.In a clinic test,CTC counting results based on the nanoprobe was more accurate,which is helpful for achieving early diagnosis of cancer.2.“Ratiometric”hydrogen peroxide responsive nanoprobe for circulating tumor cells detection in gastrointestinal cancer patients“Turn on”hydrogen peroxide responsive fluorescent nanoprobes that detect the level of hydrogen peroxide in cells through enhancement of the blue fluorescence signal are largely affected by cellular uptake of nanoprobe.Therefore,in order to reduce the influence of intracellular concentration of nanoprobes,a ratiometric hydrogen peroxide responsive fluorescent nanoprobe was designed to measure the concentration of hydrogen peroxide in tumor cells semi-quantitatively.Hydrophobic hydrogen peroxide responsive coumarin-borate probe and internal reference probe Rhodamine B were introduced to the side chains of hydrophilic glycol chitosan to prepare amphiphilic GC-Cou-Bpin-RhB graft polymers,which can self-assemble in water to form ratiometric fluorescent nanoprobes.The fluorescence intensity of Rhodamine B,an internal reference probe on the fluorescent nanoprobe,is not affected by hydrogen peroxide,whereas the coumarin-boric acid ester has a marked increase of blue fluorescent intensity when reacted with hydrogen peroxide.The GC-Cou-Bpin-RhB nanoprobe was applied to the semi-quantitative detection of hydrogen peroxide level in colon cancer cells and has been widely applied to circulating tumor cells in gastrointestinal cancer patients.Clinical experimental results demonstrated that the ratiometric hydrogen peroxide responsive fluorescent nanoprobe can effectively overcome the influence of nanoprobe concentration in cells to reflect the level of hydrogen peroxide in circulating tumor cells more accurately,and distinguish viable CTCs and apoptotic CTCs through the fluorescence of reference probe,which assist cancer patients with early tumor diagnosis.3.“Targeted”hydrogen peroxide responsive nanoprobe for circulating tumor cell identification in pancreatic cancer patientsDue to the extremely low counts of circulating tumor cells and tens of thousands of white blood cells in the whole bloodstream,the presence of a large number of white blood cells has brought a lot of interference to the detection of circulating tumor cells.Therefore,in order to improve the detection efficiency of fluorescent nanoprobes and reduce the interference of leukocytes,a CD44-targeted hydrogen peroxide responsive fluorescent nanoprobe was designed and synthesized,which can specifically detect the hydrogen peroxide in CD44-overexpressing tumor cells.A hydrophobic hydrogen peroxide responsive naphthaleneimine-boronate fluorescent molecule was grafted onto the side chains of hydrophilic hyaluronic acid in the presence of DMTMM to form amphipathic hyaluronic acid-naphthaleneimine-boronate graft polymer(HA-NP-BE),which can self-assemble into“Targeted”fluorescent nanoprobes.In vitro cell evaluation confirmed that HA-NP-BE fluorescent nanoprobe can target pancreatic cancer cells to detect intracellular hydrogen peroxide through the specific binding of hyaluronic acid and highly expressed CD44 antigen on the surface of pancreatic cancer.Clinical experiments demonstrated that HA-NP-BE fluorescent nanoprobe can detect the circulating tumor cells in pancreatic cancer patients efficiently.